The presence of multiple prion strains is a great challenge to the prion hypothesis, which postulates that the variations in the pathogenic PrP conformation, PrPSc, lead to the prion strain phenomenon. Recent advance in prion field reveals a key role of other physiological factor(s) in facilitating PrP conversion and generating prion infectivity. Our recent results showed that, in the presence of proper facilitating factors, the bacterially-expressed recombinant PrP can be converted into the infectious conformation causing bona fide prion disease in animals. This discovery strongly supports the prion hypothesis, highlights the important role of facilitating factors in prion conversion, and opens new avenues for prion research. We propose to use the de novo recombinant prion formation system to study the role of facilitating factors in enciphering prion strains. We will use the in vitro PrP conversion assay, biochemical characterization, animal bioassay, and histopathological analyses to investigate the relationship among facilitating factors, the infectious prion aggregates, and the prion strain phenomenon in the following three specific aims. In aim 1, we propose to determine whether transmission in outbred mice with a single recombinant prion preparation is capable of creating multiple prion strains. In aim 2, we will determine whether two biochemically different recombinant prions represent two different strains. In aim 3, we will test the hypothesis that distinct prion strains can be created in the presence of different facilitating factors. Results from these studies will provide us with insights into the molecular mechanism behind prion strain phenomenon, which is critically important in preventing cross-species transmission of these fatal neurodegenerative disorders.